PROJECT SUMMARY/ABSTRACT The cellular recycling process of autophagy is essential for tumor cells to survive and adapt to a variety of stresses, including ischemia and chemotherapies, which ultimately leads to more aggressive tumor growth and chemoresistance. ATG9A is known to play a governing role in driving tumor cell autophagy, yet fundamental gaps exist in our understanding of 1) how ATG9A is regulated post-transcriptionally; 2) how protein-protein interactions mediate the mobilization of ATG9A to promote autophagy; and 3) mechanisms that link defective ATG9A function to human diseases, including cancer. These gaps hinder the rational design of targeted therapies to block tumor cell autophagy. The long-term goal is to discover mechanisms of tumor cell survival and develop targeted strategies, based on these mechanisms, to exploit cancer cell vulnerabilities The overall objective of this proposal is to elucidate a detailed mechanism of ATG9A-mediated regulation of autophagy. The central hypothesis is that ATG9A phosphorylation is positively regulated by AMPK within the ULK1 complex and negatively regulated by a nutrient sensitive phosphatase, which control its mobilization and trafficking toward the autophagosome. In addition, our data revealed novel ATG9A interactions with LRBA and an ULK1-independent ATG13 subcomplex that we propose regulate ATG9A trafficking in autophagy. In particular, we posit that the interaction between ATG9A and LRBA is defective in patients carrying LRBA mutations, which may explain how these mutations cause immunodeficiency and B cell lymphoma. Guided by preliminary data, this hypothesis will be tested in the following specific aims: Aim 1: Determine the mechanism of ATG9A phosphorylation and its impact on ATG9A trafficking. Aim 2: Determine the mechanism by which an ULK1-independent ATG13 subcomplex regulates ATG9A. Aim 3: Determine if LRBA regulates ATG9A and whether disease-causing mutations in LRBA disrupt ATG9A function in autophagy. The proposal is innovative because it elucidates novel concepts/models of ATG9A regulation and applies emerging techniques (14-3-3 phospho-probing, BioID) to overcome the inherent challenges of studying ATG9A. The proposed research is significant because it fills fundamental gaps in our understanding of one of the least- understood autophagy regulators, ATG9A, and elucidates a potential role for defective ATG9A regulation in human disease.